Pathophysiological Actions of Anthrax Virulence Determinants

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications, trials & patents

Abstract

Anthrax toxin is composed of three proteins: Protective antigen (PA), lethal factor (LF) and edema factor (EF). PA carries LF and EF into cells. EF is an adenylyl cyclase converting ATP to cAMP. LF is a protease targeting cellular kinases and, uniquely, the rodent Nlrp1 inflammasome. PA binds to two receptors, capillary morphogenesis protein-2 (CMG-2) or tumor endothelial marker-8 (TEM-8). In the previous year, we reported on development of a novel mechanism for visualization of intoxication in the mouse model. Because the number of toxin molecules taken up by individual cells is too low for direct immunohistochemical visualization, functional receptors in different cell types and organs was assessed using a novel reporter system in which a fusion of the N-terminal domain of LF to Cre recombinase (LFn-Cre) was used to track uptake of LF by cells. The previous study utilized transgenic mice expressing a red fluorescent protein that converts to expression of green fluorescence when Cre is delivered by LF into cells, to assess entry in different mouse organs over time, dose and route of delivery variation tests. Intoxication at the single cell level was captured in many organs. During 2023, we extended these studies using new mouse models that have different combinations of the anthrax toxin receptors. Furthermore, in service of our labs decades long development of modified and reengineered anthrax toxins as cancer therapeutics, the methods were applied to track toxin delivery to tumor cells and host-derived vasculature in tumors. A range of recombinase responsive tumor lines which fluoresce or change fluorescence in response to recombinase delivery were developed. Tumors were tested for specific responses to various recombinases and LF-mediated delivery of Cre recombinase to tumors was visualized in mice. Work on the imaging and characterization of cellular targets of toxin therapeutics in tumors is ongoing. During 2023, we also continued long term studies using the Collaborative Cross recombinant inbred mouse collection to map genetic loci controlling unique phenotypes in response to anthrax in mice. Created from a panel of 8 unique parental founders, this mouse collection has presented new approaches to understanding anthrax toxin actions in vivo. We are in our fourth generation of mouse crosses to map genes linked to control of anthrax toxin responses.